Enhanced optical detection for nucleic acid sequencing using thermally-dependent fluorophore tags

ABSTRACT

Disclosed herein are improved methods and systems for sequencing nucleic acid that exploit the temperature-dependence of the emitted intensity of fluorescent dyes. The temperature of the sequencing reaction is adjusted during each sequencing cycle, and the emission, or lack of emission, of light meeting or exceeding a threshold by the fluorescent dyes at different temperatures, or within different temperature ranges, is used to detect the fluorescent labels of the incorporated dNTPs and thereby sequence the nucleic acid. The disclosed methods enable a determination of the dNTP incorporated at any given site with a reasonable number of chemistry steps without the complex optics necessary for prior-art systems.

BACKGROUND

Deoxyribonucleic acid (DNA) is an example of a nucleic acid. DNA is apolymer or strand composed of only four possible constituent moleculesor nucleotide bases (also referred to herein as simply “nucleotides”):adenine (A), cytosine (C), guanine (G), and thymine (T).

A DNA strand can be either single or double-stranded. Single-strandedDNA (ssDNA) can bind with its unique ssDNA complement to form adouble-stranded version of itself (dsDNA). Adenine bases bond only withthymine bases, and cytosine bases bond only with guanine bases. Forexample, the sequence ACTGGC is complementary to and bonds with TGACCG.Because bases can occur in any order, the goal of DNA sequencing is to“read” a particular ssDNA strand or template, that is, to reliablydetermine the sequence of its bases.

One type of nucleic acid sequencing used for DNA sequencing is known as“sequencing by synthesis” (SBS). SBS involves binding ofprimer-hybridized template DNA, incorporation of a deoxynucleosidetriphosphate (dNTP), and detection of incorporated dNTP. A ssDNA (i.e.,a strand to be sequenced) is sequenced through an iterative buildup ofits complement, with polymerase used to accelerate the rate ofnucleotide incorporation. In one type of SBS, a long strand of DNA to besequenced is cut into smaller strands, and these smaller strands can besequenced at the same time, potentially after being “amplified” (i.e.,copied) using a process such as bridge amplification, which is known inthe art.

In one approach to SBS, the progress of the buildup of the complement ofthe bases of a DNA strand being sequenced is inferred through thedetection of fluorescence. A fluorescent moiety is attached to each ofthe four dNTPs (A, T, C, and G). It is then possible to distinguishbetween the incorporation of different dNTPs into a growing nucleic acidstrand because each of the fluorescent moieties excites and emits lightat different wavelengths, which enables the target sequence to bedetermined. Typically, fluorescently-labeled dNTPs are excited andmeasured by one of four optical filters, one for each distinct dye, in afluorescent sequencing instrument.

Such techniques may use fluorescence to generate an image using, e.g.,an epifluorescence microscope or a confocal microscope. The specimen isilluminated with light of a specific wavelength (or wavelengths), whichis absorbed by the fluorophores, thereby causing them to emit light oflonger wavelengths (i.e., of a different color than the absorbed light).A spectral emission filter separates the illumination light from theweaker emitted fluorescence. A fluorescence microscope typicallyincludes a light source (e.g., a laser), an excitation filter, adichroic mirror (or beamsplitter), and an emission filter. The filtersand the dichroic mirror/beamsplitter are chosen to match the spectralexcitation and emission characteristics of the fluorophore used to labelthe specimen. The distribution of a single fluorophore (e.g., color,wavelength, etc.) is imaged at a time. Several single-color images mustbe combined to produce multi-color images of several types offluorophores.

State-of-the-art sequencing systems that rely on fluorescence signaldetection can provide throughputs of up to 20 billion reads per run.Achieving such performance, however, requires large-area flow cells,high-precision free-space imaging optics, and expensive high-powerlasers to generate sufficient fluorescence signals to enable successfulbase detection.

There is, therefore, an ongoing need to improve nucleic acid sequencingtechnology.

SUMMARY

This summary represents non-limiting embodiments of the disclosure.

Disclosed herein are improvements to DNA sequencing in whichfluorophores are used to tag dNTP types. The selected fluorophores emitlight in a temperature-dependent manner over a range of temperaturesbetween room temperature and an upper temperature (e.g., 100° C.). Whenthe intensity of the emitted light is below the sensitivity threshold ofthe detectors used to capture the light, the fluorophores are notdetected. Conversely, when the intensity of the emitted light is abovethe sensitivity threshold, the fluorophores are detected. By detectingthe emitted intensity (or absence of emitted intensity exceeding athreshold) at multiple different temperatures (or within multipledistinct and non-overlapping temperature ranges) a determination of thedNTP incorporated at any given site can be determined with a reasonablenumber of (e.g., fewer than conventional fluorophore-based systems)chemistry steps without the more complicated optics necessary forexisting systems.

In some embodiments, a method of sequencing nucleic acid uses asequencing apparatus comprising a fluidic channel having a plurality ofsites for attaching, to a surface of the fluidic channel, a plurality ofnucleic acid strands to be sequenced. In some such embodiments, themethod comprises, in one or more rounds of addition, adding, to thefluidic channel, (i) the plurality of nucleic acid strands, (ii) aplurality of molecules of nucleic acid polymerase, (iii) a firstfluorescently-labeled nucleotide precursor comprising a firstfluorescent label, and (iv) a second fluorescently-labeled nucleotideprecursor comprising a second fluorescent label. An intensity of thefirst fluorescent label is greater than or equal to a first threshold ina first temperature range and in a second temperature range, the secondtemperature range being lower than the first temperature range, and anintensity of the second fluorescent label is less than or equal to asecond threshold in the first temperature range and greater than orequal to the second threshold in the second temperature range. Themethod further comprises setting a temperature within the fluidicchannel to be within the first temperature range, detecting a firstintensity at each of the plurality of sites while the temperature of thefluidic channel is within the first temperature range, and in responseto the first intensity at a particular site of the plurality of sitesbeing greater than or equal to a first value, determining that the firstfluorescently-labeled nucleotide precursor has been incorporated intothe extendable primer at the particular site. The detected firstintensity at the particular site may be zero or nonzero. The methodfurther comprises setting the temperature within the fluidic channel tobe within the second temperature range, detecting a second intensity ateach of the plurality of sites while the temperature of the fluidicchannel is within the second temperature range, and in response to thesecond intensity at the particular site being greater than or equal to asecond value and the first intensity at the particular site being lessthan the first value, determining that the second fluorescently-labelednucleotide precursor has been incorporated into the extendable primer atthe particular site. The detected second intensity at the particularsite may also be zero or nonzero.

In some embodiments, the first and second thresholds are approximatelythe same. In other embodiments, the first and second thresholds aredifferent.

In some embodiments, the first and second values are approximately thesame. In other embodiments, the first and second values are different.

In some embodiments, the first value is the first threshold, and thesecond value is the second threshold. In some embodiments, the firstvalue is based on the first threshold, and the second value is based onthe second threshold.

In some embodiments, the method further comprises adding, to the fluidicchannel, a third fluorescently-labeled nucleotide precursor comprising athird fluorescent label, wherein an intensity of the third fluorescentlabel is less than a third threshold in the first and second temperatureranges and greater than or equal to the third threshold in a thirdtemperature range, the third temperature range being lower than thesecond temperature range; setting the temperature within the fluidicchannel to be within the third temperature range; detecting a thirdintensity (which may be zero or nonzero) at each of the plurality ofsites while the temperature of the fluidic channel is within the thirdtemperature range; and in response to the third intensity at theparticular site being greater than or equal to a third value, and thefirst intensity at the particular site being less than the first value,and the second intensity at the particular site being less than thesecond value, determining that the third fluorescently-labelednucleotide precursor has been incorporated into the extendable primer atthe particular site.

In some such embodiments, the first, second, and thirdfluorescently-labeled nucleotide precursors are added to the fluidicchannel at substantially the same time.

In some embodiments, the method further comprises, in response to thefirst intensity at the particular site being less than the first value,and the second intensity at the particular site being less than thesecond value, and the third intensity at the particular site being lessthan the third value, determining that a fourth, unlabeled precursor hasbeen incorporated into the extendable primer at the particular site. Insome such embodiments, at least two of the first, second, and thirdthresholds are approximately the same. In some embodiments, the firstand second thresholds are different.

In some embodiments, at least two of the first, second, and third valuesare approximately the same. In some embodiments, the first, second, andthird values are different.

In some embodiments, the method further comprises adding, to the fluidicchannel, a fourth fluorescently-labeled nucleotide precursor comprisinga fourth fluorescent label, wherein an intensity of the fourthfluorescent label is less than a fourth threshold in each of the first,second, and third temperature ranges and greater than or equal to thefourth threshold in a fourth temperature range, the fourth temperaturerange being lower than the third temperature range; setting thetemperature within the fluidic channel to be within the fourthtemperature range; detecting a fourth intensity (which may be zero ornon zero) at each of the plurality of sites while the temperature of thefluidic channel is within the fourth temperature range; and in responseto the fourth intensity at the particular site being greater than orequal to a fourth value, and the first intensity at the particular sitebeing less than the first value, and the second intensity at theparticular site being less than the second value, and the thirdintensity at the particular site being less than the third value,determining that the fourth fluorescently-labeled nucleotide precursorhas been incorporated into the extendable primer at the particular site.

In some such embodiments, two or more of the first, second, third, andfourth thresholds are approximately the same.

In some embodiments, two or more of the first, second, third, and fourthvalues are approximately the same.

In some embodiments, the first, second, third, and fourthfluorescently-labeled nucleotide precursors are added to the fluidicchannel at substantially the same time.

In some embodiments, a method of sequencing nucleic acid using asequencing apparatus comprising a fluidic channel having a plurality ofsites for attaching, to a surface of the fluidic channel, a plurality ofnucleic acid strands to be sequenced, comprises: in one or more roundsof addition, adding, to the fluidic channel, (i) the plurality ofnucleic acid strands, (ii) a plurality of molecules of nucleic acidpolymerase, (iii) a first fluorescently-labeled nucleotide precursorcomprising a first fluorescent label, wherein an intensity of the firstfluorescent label is less than a first threshold in a first temperaturerange and greater than or equal to the first threshold in a secondtemperature range, the second temperature range being lower than thefirst temperature range, (iv) a second fluorescently-labeled nucleotideprecursor comprising a second fluorescent label, wherein an intensity ofthe second fluorescent label is greater than or equal to a secondthreshold in the first temperature range and in the second temperaturerange, and (v) a third fluorescently-labeled nucleotide precursorcomprising the first and second fluorescent labels; setting atemperature within the fluidic channel to be within the firsttemperature range; detecting a first intensity (which may be zero ornonzero) at each of the plurality of sites while the temperature of thefluidic channel is within the first temperature range; setting thetemperature within the fluidic channel to be within the secondtemperature range; detecting a second intensity (which may be zero ornonzero) at each of the plurality of sites while the temperature of thefluidic channel is within the second temperature range; and determiningwhether one of the first, second, or third fluorescently-labelednucleotide precursors has been incorporated into the extendable primerat a particular site of the plurality of sites.

In some such embodiments, the first, second, and thirdfluorescently-labeled nucleotide precursors are added to the fluidicchannel at substantially the same time.

In some embodiments, determining whether one of the first, second, orthird fluorescently-labeled nucleotide precursors has been incorporatedinto the extendable primer at the particular site of the plurality ofsites comprises: in response to the first intensity at the particularsite being less than a first value, and the second intensity at theparticular site being greater than or equal to a second value,determining that the first fluorescently-labeled nucleotide precursorhas been incorporated into the extendable primer at the particular site;in response to the first intensity at the particular site being greaterthan or equal to the first value, and the second intensity at theparticular site being greater than or equal to a third value, the thirdvalue being greater than each of the first and second values,determining that the third fluorescently-labeled nucleotide precursorhas been incorporated into the extendable primer at the particular site;and in response to the first intensity at the particular site beinggreater than or equal to the first value, and the second intensity atthe particular site being greater than or equal to the second value andless than the third value, determining that the secondfluorescently-labeled nucleotide precursor has been incorporated intothe extendable primer at the particular site.

In some such embodiments, the method further comprises, in response tothe first intensity at the particular site being less than the firstvalue, and the second intensity at the particular site being less thanthe second value, determining that a fourth, unlabeled nucleotideprecursor has been incorporated into the extendable primer at theparticular site.

In some embodiments, the method further comprises determining that noneof the first, second, or third fluorescently-labeled nucleotideprecursors has been incorporated into the extendable primer at theparticular site, and, in response to determining that none of the first,second, or third fluorescently-labeled nucleotide precursors has beenincorporated into the extendable primer at the particular site,inferring that a fourth, unlabeled nucleotide precursor has beenincorporated into the extendable primer at the particular site.

In some embodiments, the first and second thresholds are approximatelythe same. In other embodiments, the first and second thresholds aredifferent.

In some embodiments, the first and second values are substantially thesame. In other embodiments, the first and second values are different.

In some embodiments, the third value is approximately or exactly the sumof the first and second values.

In some embodiments, the first value is the first threshold, and thesecond value is the second threshold.

In some embodiments, the first value is based on the first threshold,and the second value is based on the second threshold.

In some embodiments, the third value is approximately the sum of thefirst and second values.

In some embodiments, a system for sequencing nucleic acid comprises afluidic channel, a heater coupled to the fluidic channel, an imagingsystem, and at least one processor. In some embodiments, the fluidicchannel has a plurality of sites for attaching, to a surface of thefluidic channel, a plurality of nucleic acid strands to be sequenced. Insome embodiments, the heater is configured to set a temperature of acontents of the fluidic channel to be within any of nonoverlappingfirst, second, third, and fourth temperature ranges. In someembodiments, the imaging system is configured to detect an intensity ateach of the plurality of sites in each of the first, second, third, andfourth temperature ranges. In some embodiments, the second temperaturerange is lower than the first temperature range, the third temperaturerange is lower than the second temperature range, and the fourthtemperature range is lower than the third temperature range.

The at least one processor is configured to execute at least onemachine-readable instruction. In some embodiments, when executed, the atleast one machine-executable instruction causes the at least oneprocessor to identify a first fluorescently-labeled nucleotide precursoror a complementary base of the first fluorescently-labeled nucleotideprecursor in response to an intensity at a particular site of theplurality of sites being greater than or equal to a first threshold inthe first temperature range and in the second temperature range. In someembodiments, when executed, the at least one machine-executableinstruction causes the at least one processor to identify a secondfluorescently-labeled nucleotide precursor or a complementary base ofthe second fluorescently-labeled nucleotide precursor in response to theintensity at the particular site of the plurality of sites being lessthan a second threshold in the first temperature range and greater thanor equal to the second threshold in the second temperature range. Insome embodiments, when executed, the at least one machine-executableinstruction causes the at least one processor to identify a thirdfluorescently-labeled nucleotide precursor or a complementary base ofthe third fluorescently-labeled nucleotide precursor in response to theintensity at the particular site of the plurality of sites being lessthan a third threshold in the first and second temperature ranges andgreater than or equal to the third threshold in the third temperaturerange. In some embodiments, when executed, the at least onemachine-executable instruction causes the at least one processor toidentify a fourth fluorescently-labeled nucleotide precursor or acomplementary base of the fourth fluorescently-labeled nucleotideprecursor in response to the intensity at the particular site of theplurality of sites being less than a fourth threshold in each of thefirst, second, and third temperature ranges and greater than or equal toa fourth threshold in the fourth temperature range.

In some embodiments, the fluidic channel comprises a structure (e.g., acavity, a ridge, etc.) that includes the plurality of sites forattaching, to the surface of the fluidic channel, the plurality ofnucleic acid strands to be sequenced.

In some embodiments, the heater comprises a thermal sensor or amicroprocessor.

In some embodiments, the imaging system comprises a camera, a lightsource (e.g., a laser), illumination optics, a detector, an opticalblocking filter, an analog-to-digital converter (ADC), or one or moresensors. The illumination optics may be configured to distribute theexcitation light substantially uniformly over the plurality of sites. Insome embodiments, the detector comprises a lens. In some suchembodiments, the imaging system includes an optical blocking filter thatis situated within the system such that it substantially isolates thelens from light emitted by the light source. In some embodiments, theone or more sensors of the imaging system comprise a charge-coupleddevice (CCD) sensor.

In some embodiments, when executed by the at least one processor, the atleast one machine-readable instruction further causes the at least oneprocessor to control the heater, the imaging system, or both.

In some embodiments, a system for sequencing nucleic acid comprises afluidic channel, a heater coupled to the fluidic channel, an imagingsystem, and at least one processor. In some embodiments, the fluidicchannel has a plurality of sites for attaching, to a surface of thefluidic channel, a plurality of nucleic acid strands to be sequenced. Insome embodiments, the heater is configured to set a temperature of acontents of the fluidic channel to be within either of first and secondtemperature ranges, wherein the first and second temperature ranges arenonoverlapping. In some embodiments, the imaging system is configured todetect an intensity at each of the plurality of sites in each of thefirst and second temperature ranges.

The at least one processor is configured to execute at least onemachine-readable instruction. In some embodiments, when executed, the atleast one machine-executable instruction causes the at least oneprocessor to instruct the heater to set a temperature within the fluidicchannel to be within the first temperature range and obtain, from theimaging system, an indication of a first intensity at each of theplurality of sites while the temperature of the fluidic channel iswithin the first temperature range. In some embodiments, when executed,the at least one machine-executable instruction further causes the atleast one processor to instruct the heater to set the temperature withinthe fluidic channel to be within the second temperature range andobtain, from the imaging system, an indication of a second intensity ateach of the plurality of sites while the temperature of the fluidicchannel is within the second temperature range. In some embodiments,when executed, the at least one machine-executable instruction furthercauses the at least one processor to determine that a firstfluorescently-labeled nucleotide precursor has been incorporated into anextendable primer at the particular site in response to the firstintensity at a particular site of the plurality of sites being less thana first value, and the second intensity at the particular site beinggreater than or equal to a second value. In some embodiments, whenexecuted, the at least one machine-executable instruction further causesthe at least one processor to determine that a secondfluorescently-labeled nucleotide precursor has been incorporated intothe extendable primer at the particular site in response to the firstintensity at the particular site being greater than or equal to thefirst value, and the second intensity at the particular site beinggreater than or equal to the second value and less than a third value,the third value being greater than each of the first and second values.In some embodiments, when executed, the at least one machine-executableinstruction further causes the at least one processor to determine thata third fluorescently-labeled nucleotide precursor has been incorporatedinto the extendable primer at the particular site in response to thefirst intensity at the particular site being greater than or equal tothe first value, and the second intensity at the particular site beinggreater than or equal to the third value.

BRIEF DESCRIPTION OF THE DRAWINGS

Objects, features, and advantages of the disclosure will be readilyapparent from the following description of certain embodiments taken inconjunction with the accompanying drawings in which:

FIG. 1A illustrates the temperature dependence of the fluorescentintensity of a fluorescent dye.

FIG. 1B illustrates the temperature dependence of the fluorescentintensities of additional fluorescent dyes.

FIG. 1C is an annotated version of FIG. 1B showing how the temperaturedependence of the emissions of the four fluorescent dyes can beexploited for nucleic acid sequencing.

FIG. 2A illustrates the use of four intensity-measuring events, fourtemperature ranges, and four fluorescently-labeled nucleotide precursorsfor nucleic acid sequencing in accordance with some embodiments.

FIG. 2B illustrates conceptually the use of three intensity-measuringevents, three temperature ranges, and three fluorescently-labelednucleotide precursors, each having a different fluorescent label, inaccordance with some embodiments.

FIG. 2C illustrates conceptually the use of two intensity-measuringevents, two temperature ranges, and two fluorescently-labeled nucleotideprecursors, each having a different fluorescent label, in accordancewith some embodiments.

FIG. 3A is a flowchart illustrating a method of sequencing nucleic acidin accordance with some embodiments.

FIG. 3B illustrates how the method can continue after FIG. 3A inaccordance with some embodiments.

FIG. 3C illustrates another way the method can continue after FIG. 3A inaccordance with some embodiments.

FIG. 3D illustrates an alternative ordering of elements of the methodillustrated in FIG. 3A in accordance with some embodiments.

FIG. 3E illustrates another alternative ordering of elements of themethod illustrated in FIG. 3A in accordance with some embodiments.

FIGS. 4A and 4B illustrate a method of sequencing nucleic acid inaccordance with some embodiments.

FIG. 5 is a block diagram of a system for sequencing nucleic acid inaccordance with some embodiments.

FIG. 6 is a block diagram of components of an imaging system that may beused in accordance with some embodiments.

DETAILED DESCRIPTION

The term “nucleotide” as used herein includes both deoxyribonucleotidesand ribonucleotides, as well as modified nucleotides such asdideoxynucleotides and other chain terminator nucleotides. It alsoincludes nucleotide residues within a polynucleotide. The terms“nucleotide” and “base” are used interchangeably herein.

The term “fluorescent dye” as used herein refers to a dye whose presencecan be detected by fluorescence. In particular embodiments, thefluorescent dyes used herein are suitable for nucleic acid sequencing.

The term “nucleic acid polymerase” includes DNA polymerases and RNApolymerases. The DNA polymerases can be DNA-dependent or RNA-dependent(i.e., reverse transcriptase). DNA polymerases include, for example, TaqDNA polymerase (Thermus aquaticus DNA polymerase), mutant forms of TaqDNA polymerase, such as Taq G46D F667Y DNA polymerase, and others knownin the art.

The term “nucleic acid substrate” as used herein refers to a nucleicacid that is modified by a chemical or enzymatic reaction, or to anucleic acid that serves as a template for a chemical or enzymaticreaction. For instance, in a nucleic acid sequencing reaction, both theoligonucleotide that is extended by a polymerase and the templatenucleic acid are “nucleic acid substrate,” as the term is used herein.

As used herein, the term “chain terminator nucleotide” refers to amononucleotide that can be incorporated into a growing polynucleotidechain by a nucleic acid polymerase and that when incorporated terminatesthe chain because a polynucleotide containing the chain terminatornucleotide at its 3′ terminus cannot serve as a substrate for theaddition of another nucleotide by the nucleic acid polymerase. Typicalterminators are those in which the nucleobase is a purine, a7-deaza-purine, a pyrimidine, a normal nucleobase or a common analogthereof and the sugar moiety is a pentose that includes a 3′-substituentthat blocks further synthesis, such as a dideoxynucleotide (ddNTP).Examples of ddNTPs are ddGTP, ddATP, ddTTP, and ddCTP. Substituents thatblock further synthesis include, but are not limited to, amino, deoxy,halogen, alkoxy and aryloxy groups. Exemplary terminators include, butare not limited to, those in which the sugar-phosphate ester moiety is3′-(C₁-C₆)alkylribose-5′-triphosphate,2′-deoxy-3′-(C₁-C₆)alkylribose-5′-triphosphate,2′-deoxy-3′-(C₁-C₆)alkoxyribose-5′-triphosphate,2′-deoxy-3′-(C₅-C₁₄)aryloxyribose-5′-triphosphate,2′-deoxy-3′-haloribose-5′-triphosphate,2′-deoxy-3′-aminoribose-5′-triphosphate,2′,3′-dideoxyribose-5′-triphosphate or2′,3′-didehydroribose-5′-triphosphate.

Existing fluorescence-based technologies used to differentiate betweendifferent bases in a sample (e.g., in fluorescence-based nucleic acidsequencing technologies) rely on, for example, the quality of a signalgenerated by a detection moiety that is associated with a particulartype of nucleotide. For example, traditional fluorescent sequencingtechnologies utilize identifiably distinct fluorescent moieties, eachattached to one of the four nucleotides A, T, C, and G that are utilizedin a sequencing reaction.

One conventional method of DNA sequencing involves adapting ssDNA forattachment to a solid support of a sequencing apparatus and amplifyingthe quantity of the ssDNA using techniques such as the polymerase chainreaction to create many DNA molecules with a short leader. An oligocomplementary to the short leader may then be added so that there is ashort section of dsDNA at the leader. The double stranded portion of thebound molecule is a primer for a suitable DNA polymerase, such as, forexample, Taq polymerase, which is operable at high temperatures.

The sequencing can then take one of several approaches. For example, thesequencing can use a mixture of four fluorescently-labeled 3′-blockedNTPs (fluorescently labeled dideoxynucleotide terminators), where thefluorescent label is part of the 3′-blocking group. The fluorescentlabel serves as a “reversible terminator” for polymerization. Each ofthe NTPs is labeled by a different label (i.e., each of the A, G, C, andT nucleotides has a different label), and the different labels aredistinguishable by fluorescent spectroscopy or by other optical means.

Four fluorescently-labeled nucleotide precursors can be used to sequencemillions of clusters of DNA strands in parallel. DNA polymerasecatalyzes the incorporation of fluorescently-labeled dNTPs into a DNAtemplate strand during sequential cycles of DNA synthesis. In eachsequencing cycle, the bound double strand DNA molecule is exposed to DNApolymerase and a mixture of the four fluorescently-labeled 3′-blockedNTPs. The polymerase adds one of the four dNTPs to the growingoligonucleotide chain (i.e., whichever dNTP is complementary to the nextunpaired base in the ssDNA). The unincorporated dNTPs and otherimpurities that are either left unreacted or generated during thereactions are then separated from the vicinity of the support-bound DNAby washing at a temperature that prevents the free dNTPs from binding tothe ssDNA but is not so high as to dehybridize the dsDNA.

Because only one of the four types of dNTP will have been added to theoligonucleotide, and the four fluorescent labels are distinguishable,the identity of the incorporated dNTP can be identified through laserexcitation and imaging. Specifically, each of four filters is used todetermine whether light of a particular wavelength (e.g., color) isemitted. The fluorescent label can then be enzymatically cleaved toallow the next round of incorporation. Because each base type can pairwith one and only one other base type, the identity of the just-pairedbase in the unknown sequence of the ssDNA is known from the identity ofthe incorporated dNTP (which is known from the wavelength of emittedlight). Thus, the base is identified directly from fluorescencemeasurements during each cycle.

One disadvantage of the above-described approach is that a complicatedoptics system is needed to filter out different wavelengths of light todetect the fluorescent labels of the incorporated dNTPs and todistinguish between the different emitted colors. Other approaches havebeen developed to simplify the optics system, but they are slower tosequence and require intermediate chemistry steps within each sequencingcycle. Thus, these approaches have been introduced in smaller, lessexpensive entry-level sequencing systems but not in higher-level systemsrequiring fast throughput.

Disclosed herein are improved methods of sequencing nucleic acid. Theinventors had the insight that some fluorescent dyes emit light at anintensity that varies with temperature, and this temperature-dependenceof the emitted intensity can be exploited for nucleic acid sequencingapplications. The disclosed methods enable the use of simpler opticssystems than the conventional four-dye approach described above becausethey do not require wavelength filtering. Instead, the temperature ofthe system is adjusted during each sequencing cycle (e.g., by using anintegrated heating element of the sequencing device). Furthermore, allfour bases may be introduced in a single step. The emission, or lack ofemission, of light by the fluorescent dyes at different temperatures (orwithin different temperature ranges) can be used to detect thefluorescent labels of the incorporated dNTPs and thereby sequence thenucleic acid. For instance, and as will be described in further detailbelow in the context of some embodiments, in a composition containingfour chain terminator nucleotides, each linked to a differentfluorescent dye, each of the four fluorescent dyes will emit light in atemperature-dependent manner such that it is distinguishable from theother dyes.

The temperature response of fluorescent dyes has been used to performmicroscale local temperature measurements of microfluidic and biologicalsystems. Some such systems are described in the 2001 paper entitled“Temperature Measurement in Microfluidic Systems Using aTemperature-Dependent Fluorescent Dye” by D. Ross, M. Gaitan, and L.Locasco (Analytical Chemistry, Vol. 73, No. 17, Sep. 1, 2001). FIG. 1Aherein illustrates the temperature dependence of the fluorescenceintensity of one such fluorescent dye, namely Rhodamine B. As shown inFIG. 1A, the intensity decreases monotonically as the temperatureincreases. Above room temperature (assumed to be 20 degrees Celsius),the intensity of emitted light decreases rapidly. Thus, if a detector ina nucleic acid sequencing apparatus were to have a detection thresholdof, for example 80% of the peak intensity, it would detect the dye attemperatures below about 32 degrees Celsius but not above thattemperature.

For comparison, FIG. 1B illustrates the temperature dependence of thefluorescent intensities of various coumarin dyes, namely4-methyl-7-methoxy (labeled “I” in FIG. 1B), 4-methyl-5,7-diethoxy(labeled “II”), 4-methyl-5-ethoxy-7-methoxy (labeled “III”), and4-methyl-7,8-diethoxy (labeled “IV”). (See, e.g., R. Giri, “Temperatureeffect study upon the fluorescence emission of substituted coumarins,”Spectrochimica Acta Part A: Molecular Spectroscopy, Volume 48, Issue 6,June 1992, p. 843-848.) As FIG. 1B shows, the intensity of4-methyl-7,8-diethoxy (IV) is relatively insensitive to temperature. Incontrast, the intensity of 4-methyl-7-methoxy (I) decreases rapidly withincreasing temperature.

Thus, the inventors had the insight that the intensity response can beexploited so that two different fluorescent labels with differentresponses to changes in temperature can be used to distinguish betweentwo nucleotides in nucleic acid sequencing. For example, a comparison ofthe temperature responses of Rhodamine (FIG. 1A) and coumarin (III)(FIG. 1B) indicates that at 50 degrees Celsius, the intensity ofRhodamine is half of its value at room temperature, whereas theintensity of coumarin (III) is ninety percent of its value at roomtemperature. Assuming the intensities of Rhodamine and coumarin (III)are approximately the same at room temperature, by tuning aphotodetector to have a minimum sensitivity for light intensity set to avalue between 50% and 90% (e.g., 75%), the photodetector would detectlight from both dyes at room temperature but only for coumarin (III) at50 degrees Celsius. One advantage of this approach relative toconventional approaches to nucleic acid sequencing is that there is noneed to filter the wavelength of emitted light or determine the color oflight emitted by the dye; knowledge of the temperature-dependence of theintensity of light emitted by the dye when excited into fluorescenceallows the different dyes to be distinguished. Therefore, the need forthe complexities and expense associated with a need to have opticalfilters is reduced or eliminated altogether.

It is to be understood that the intensity profiles of the selected dyesneed not be monotonic, smooth, or linear. It is contemplated that theselected dyes may have predictable emitted intensities in sometemperature ranges and unpredictable (or nonlinear, random, etc.)emitted intensities in other ranges.

FIG. 1C is an annotated version of FIG. 1B to illustrate how thetemperature-dependence of different fluorescent dyes can be used todistinguish between the dyes. It is to be understood that FIGS. 1B and1C show the normalized intensities of the four fluorescent dyes. Thus,this explanation assumes that the peak intensity of light emitted byeach of the fluorescent dyes at room temperature (approximately 273degrees Kelvin if 20 degrees Celsius is room temperature) isapproximately the same.

As shown in FIG. 1C, a threshold of approximately 85% intensity has beenselected as an example. In the highest temperature range, labeled “Range1,” only the dye IV emits light at an intensity that meets or exceedsthe threshold. In the next-highest temperature range, labeled “Range 2,”only the dyes III and IV emit light at an intensity that meets orexceeds the threshold. In the next-highest temperature range, labeled“Range 3,” lowest temperature range, only the dyes II, III, and IV emitlight at an intensity that meets or exceeds the threshold. Finally, inthe lowest temperature range, labeled “Range 4,” all four of thefluorescent dyes emit light at an intensity that meets or exceeds thethreshold.

Continuing with the example shown in FIG. 1C, in a DNA sequencingapplication, each of the four nucleotides can be labeled with adifferent one of the four fluorescent dyes shown. For example, A can belabeled using dye I, G can be labeled using dye II, T can be labeledusing dye III, and C can be labeled using dye IV. When the temperatureis within Range 1, only dye IV will emit light at an intensity thatmeets or exceeds the threshold. Thus, if, in the temperature Range 1,light is detected at or above the threshold, the incorporated base mustbe cytosine (C). On the other hand, if, in the temperature Range 1, nolight is detected at or above the threshold, the incorporated base isnot cytosine (C). An image may be taken in each of the four temperatureranges to record the detected light (or absence of detected light). Asused herein, the term “image” refers to any record of the presence orabsence of emitted light (e.g., an optical response over a plurality ofphysical locations). For example, an image may be, literally, an imageof a sequencing apparatus's fluidic channel, recorded using a camera,potentially using a filter to detect light of a particular wavelength atparticular physical locations where nucleic acid strands (or portions ofstrands) are attached or bound. As another example, an image may be adata file recording the detected intensity of emitted light (e.g., incandelas, lumens or any other suitable units) at various physicallocations where nucleic acid is attached or bound. As yet anotherexample, an image may be a data file recording whether or not light wasdetected at a particular location where nucleic acid is attached orbound (e.g., a 1 if light exceeding a threshold was detected, a 0 iflight exceeding the threshold was not detected). Whatever its form, animage may be stored temporarily or permanently (e.g., so its contentsmay be compared to other images), or it may be transient (e.g., createdand compared in real time to a previously-created-and-stored image).

When the temperature is within Range 2, only dyes III (T) and IV (C)will emit light at an intensity that meets or exceeds the threshold.Thus, if, in the temperature Range 2, light is detected at or above thethreshold, the incorporated base is not adenine (A) or guanine (G).Based on knowledge of whether light above the threshold is detected whenthe temperature is within Range 1, it can be determined whether theincorporated base is cytosine (C) (light detected in Range 1 and Range2) or thymine (T) (no light detected in Range 1, light detected in Range2).

When the temperature is within Range 3, all of the dyes except for dye Iwill emit light at an intensity above a threshold. Thus, if in thetemperature Range 3, light is detected at or above the threshold, it canbe concluded that the incorporated base is not adenine (A). Based onknowledge of whether light above the threshold is detected when thetemperature is within Range 1, within Range 2, and within Range 3 it canbe determined whether the incorporated base is cytosine (C) (lightdetected in Ranges, 1, 2, and 3), thymine (T) (no light detected inRange 1, but light detected in Ranges 2 and 3) or guanine (G) (no lightdetected in Ranges 1 and 2, but light detected in Range 3).

When the temperature is within Range 4, all of the dyes will emit lightat or above a threshold. Based on an assessment of the detection oflight (or absence of light) in each of the four temperature ranges, itcan be determined which nucleotide has been incorporated in thesequencing step. Specifically, the nucleotide is cytosine (C) if lightis detected in all four temperature ranges; the nucleotide is thymine(T) if light is detected only in Ranges 2, 3, and 4; the nucleotide isguanine (G) if light is detected only in Ranges 3 and 4; and thenucleotide is adenine (A) if light is detected only in Range 4.

As this example illustrates, by manipulating the temperature during eachsequencing step (i.e., setting the temperature to different valuesduring a sequencing cycle and detecting the presence or absence ofemitted light at or above a threshold), the different incorporated basescan be distinguished from one another. The temperature ranges can betested in any order. It will be appreciated that when it is desirable todetermine whether and which base has been incorporated in a particularstrand of nucleic acid being sequenced, beginning in Range 1 may beadvantageous because if light is detected in Range 1 during a cycle, itcan be concluded that the incorporated nucleotide is cytosine (C). Thus,there is no need to check Ranges 2, 3 or 4 if emitted light at or abovea threshold is detected in Range 1. It will also be appreciated thattesting in Range 3 first may be advantageous because if no light isdetected in Range 3, it can be concluded that the incorporatednucleotide is adenine (A). Thus, there is no need to check Ranges 1, 2,or 4.

As described previously, in some types of SBS, a long strand of DNA is(or a plurality of long strands of DNA from a single donor organism are)cut into smaller, random-length segments prior to sequencing. All ofthese smaller strands, which are from the same donor, are randomizedsub-strands of the complete strand to be sequenced. For example, if thecomplete strand includes the sequence ATGGCTTAGCCATACCGAT, the smallerstrands could include, for example, distinct sub-strands (e.g., TGGCTTand CCATACCGA) as well as, if a plurality of the longer strands are cutinto sub-strands, sub-strands that partially or completely overlap othersub-strands (e.g., CTTAGCCAT and ATGGCTTAGCC). All of the smaller,randomized sub-strands may be sequenced at the same time, potentiallyafter being amplified. In such applications, it will be appreciated thatbecause the sub-strands do not represent the same sub-sequences, it isdesirable to determine the intensity in each of the temperature rangesbecause the sequencing of the sub-strands will not be coordinated (orsynchronized) amongst sub-strands. For example, during a singlesequencing cycle, a first sub-strand may incorporate cytosine, a secondsub-strand might incorporate thymine, and a third sub-strand mightincorporate adenine. In order to sequence multiple random segments of alarger nucleic acid strand, it is desirable, in each sequencing cycle,to determine whether and at which physical location(s) each dNTP hasbeen incorporated.

As will be understood by those having skill in the art in view of thedisclosures herein, the same general procedure as described in thecontext of FIG. 1C can be used if the peak intensities of the differentdyes differ. In such cases, the intensity threshold applicable withinthe different temperature ranges may differ, and the appropriate valuesmay be determined based on knowledge of the temperature-dependentcharacteristics of each of the fluorescent dyes.

FIGS. 2A through 2C illustrate how the different incorporatednucleotides can be identified using these principles in accordance withsome embodiments. Each of the four nucleotides can be labeled with adifferent dye, each dye having a different fluorescence intensitydependence on temperature. The colors of the dyes are immaterial, aslong as the temperature at which the intensity of the fluorescence dropsbelow a threshold is different for each dye.

FIG. 2A illustrates conceptually the use of four images, fourtemperature ranges, and four fluorescently-labeled nucleotideprecursors, each having a different fluorescent label (which may becleavable), in accordance with some embodiments. FIG. 2A is effectivelya logic table that can be applied individually to determine, for eachnucleic acid strand or sub-strand, which nucleotide has beenincorporated into that strand or sub-strand. For example, if thefluorescent dyes, threshold, and temperature ranges shown in FIG. 1C areassumed, A is labeled using dye I, G is labeled using dye II, T islabeled using dye III, and C is labeled using dye IV. Followingincorporation of a nucleotide into a growing nucleic acid chain, thetemperature of the reaction is set to be within one of the temperatureranges. For purposes of this explanation, assume that the testingproceeds from the highest temperature to the lowest temperature. Asexplained above, this order is arbitrary, and the temperature ranges canbe tested in any order. Moreover, depending on the results obtained whenthe temperature is within a particular range, it may be unnecessary in asequencing cycle to test more than one temperature range. As explainedabove, when randomized sub-strands are sequenced at the same time, itwill be appreciated that because the sub-strands do not represent thesame sub-sequences, it is desirable to determine the intensity in eachof the temperature ranges because the sequencing of the sub-strands willnot be coordinated (or synchronized) amongst sub-strands.

After the temperature is set to be within Range 1, which, in thisexample, is assumed to be the highest of the four temperature ranges,the reaction is then exposed to light, and fluorescence is observed andrecorded as Image 1; this constitutes a first imaging event and a firstfluorescence detection pattern. The temperature is then decreased to bewithin Range 2. The reaction location is once again illuminated and thefluorescence is captured and recorded as Image 2, constituting a secondimaging event (i.e., a second fluorescence detection pattern). Thetemperature is then decreased to be within Range 3. The reactionlocation is once again illuminated and the fluorescence is captured andrecorded as Image 3, constituting a third imaging event (i.e., a thirdfluorescence detection pattern). The temperature is then decreased to bewithin Range 4. The reaction location is once again illuminated and anychange in fluorescence is captured and recorded, constituting a fourthimaging event (i.e., a fourth fluorescence detection pattern).

As denoted in FIG. 2A, Image 1 is taken when the temperature is withinRange 1, Image 2 is taken when the temperature is within Range 2, Image3 is taken when the temperature is within Range 3, and Image 4 is takenwhen the temperature is within Range 4. Within the temperature Range 4,all four dyes emit light at an intensity greater than the threshold, asshown in FIG. 2A, and therefore it is not possible to determine fromImage 4 alone which of the four fluorescently-labeled nucleotideprecursors has been incorporated into the extendible primer. Within thetemperature Range 3, however, only the fluorescent dyes labeling G, T,and C will emit light at an intensity greater than the threshold.Therefore, based on the presence of emitted light ofgreater-than-threshold intensity in Image 4 and the absence of emittedlight at or above a threshold in Image 3, one can determine that theincorporated nucleotide precursor is A. Similarly, only the fluorescentdyes labeling T and C will emit light at an intensity greater than thethreshold within the temperature Range 2. Therefore, based on thepresence of emitted light of greater-than-threshold intensity in Images4 and 3 and the absence of emitted light at or above a threshold inImage 2, one can determine that the incorporated nucleotide precursor isG. Similarly, only the fluorescent dye labeling C will emit light at anintensity greater than the threshold within the temperature Range 1.Therefore, based on the presence of emitted light ofgreater-than-threshold intensity in Images 4, 3, and 2 and the absenceof emitted light at or above a threshold in Image 1, one can determinethat the incorporated nucleotide precursor is T. And, of course, if thedetected intensity exceeds the threshold in all four of the images, itcan be determined that the incorporated nucleotide precursor is C.

Because all four of the fluorescently-labeled nucleotide precursors emitlight at an intensity meeting or exceeding the threshold in thetemperature Range 4, it is possible to simplify the procedure byeliminating imaging in temperature Range 4. FIG. 2B illustratesconceptually the use of three images, three temperature ranges, andthree fluorescently-labeled nucleotide precursors, each having adifferent fluorescent label, in accordance with some embodiments. LikeFIG. 2A, FIG. 2B is effectively a logic table that can be appliedindividually to determine, for each nucleic acid strand or sub-strand,which nucleotide has been incorporated into that strand or sub-strand.The fourth nucleotide precursor (shown as guanine (G) in FIG. 2B) isunlabeled. In this approach, Image 1 is taken while the temperature isin Range 1, Image 2 is taken while the temperature is in Range 2, andImage 3 is taken while the temperature is in Range 3.

The fluorescent dye labeling cytosine (C) is the only dye that will emitlight at an intensity greater than or equal to the threshold withintemperature Range 1. Thus, if the detected intensity in Image 1 isgreater than or equal to the threshold, it can be determined that theincorporated nucleotide precursor is cytosine (C). Otherwise, Image 2can be used to determine whether the incorporated nucleotide precursoris T: because only the fluorescent dyes labeling thymine (T) andcytosine (C) will emit light at an intensity greater than the thresholdwithin the temperature Range 2, the presence of emitted light ofgreater-than-threshold intensity in Image 2 and less-than-thresholdintensity in Image 1 indicates that the incorporated nucleotideprecursor is thymine (T). Similarly, the fluorescent dye labelingadenine (A) will emit light at an intensity greater than the thresholdonly within Range 3. Therefore, based on the presence of emitted lightof greater-than-threshold intensity in Image 3, but not in Image 2 orImage 1, one can determine that the incorporated nucleotide precursor isadenine (A). If emitted light of greater-than-threshold intensity is notdetected in any of Images 1, 2, or 3, it can be concluded (e.g.,inferred) that the incorporated nucleotide is guanine (G).

FIG. 2C illustrates an alternative embodiment in which only two imagesare used to determine which of the four nucleotide precursors has beenincorporated, in accordance with some embodiments. This embodiment usesa photodetector that is intensity-sensitive above its intensitythreshold, which means it not only detects that emitted light is presentbut also detects the intensity of the emitted light. Similarly to FIGS.2A and 2B, FIG. 2C is effectively a logic table that can be appliedindividually to determine, for each nucleic acid strand or sub-strand,which nucleotide has been incorporated into that strand or sub-strand.One nucleotide (shown as T in FIG. 2C) is labeled with a first dye, anda second nucleotide (shown as C in FIG. 2C) is labeled with a seconddye. One nucleotide (shown as A in FIG. 2C) is labeled with both dyes.The fourth nucleotide (shown as G in FIG. 2C) is unlabeled.

There are a number of ways to attach and, when necessary, cleave thefluorescent labels. For example, the fluorescent labels may be attachedto a base, in which case they may be cleaved chemically. As anotherexample, the fluorescent labels may be attached to a phosphate, in whichcase they may be cleaved by polymerase or, if attached via a linker, bycleaving the linker.

In some embodiments, the fluorescent label is linked to the nitrogenousbase (A, C, T, G, or a derivative) of the nucleotide precursor. Afterincorporation of the nucleotide precursor and the detection of theemitted light, the fluorescent label is cleaved from the incorporatednucleotide.

In some embodiments, the fluorescent label is attached via a cleavablelinker. Cleavable linkers are known in the art and have been described,e.g., in U.S. Pat. Nos. 7,057,026, 7,414,116 and continuations andimprovements thereof. In some embodiments, the fluorescent label isattached to the 5-position in pyrimidines or the 7-position in purinesvia a linker comprising an allyl or azido group. In other embodiments,the linker comprises a disulfide, indole, a Sieber group, a t-butylSieber group, or dialkoxybenzyl group. The linker may further containone or more substituents selected from alkyl (C₁₋₆) or alkoxy (C₁₋₆),nitro, cyano, fluoro groups or groups with similar properties. Briefly,the linker can be cleaved by water-soluble phosphines or phosphine-basedtransition metal-containing catalysts. Other linkers and linker cleavagemechanisms are known in the art. For example, linkers comprising tritylgroups, p-alkoxybenzyl ester groups, p-alkoxybenzyl amides groups,tert-butyloxycarbonyl (Boc) groups, and acetal based groups can becleaved under acidic conditions by a proton-releasing cleavage agent,such as an acid. A thioacetal or other sulfur-containing linker can becleaved using a thiophilic metals, such as nickel, silver or mercury.The cleavage protecting groups can also be considered for thepreparation of suitable linker molecules. Ester- and disulfidecontaining linkers can be cleaved under reductive conditions. Linkerscontaining triisopropyl silane (TIPS) or t-butyldimethyl silane (TBDMS)can be cleaved in the presence of F ions. Photocleavable linkers cleavedby a wavelength that does not affect other components of the reactionmixture include linkers comprising o-nitrobenzyl groups. Linkerscomprising benzyloxycarbonyl groups can be cleaved by Pd-basedcatalysts.

In some embodiments, the nucleotide precursor comprises a fluorescentlabel attached to a polyphosphate moiety as described in, e.g., U.S.Pat. Nos. 7,405,281 and 8,058,031. Briefly, the nucleotide precursorcomprises a nucleoside moiety and a chain of 3 or more phosphate groupswhere one or more of the oxygen atoms are optionally substituted, e.g.,with S. The label may be attached to the α, β, γ or higher phosphategroup (if present) directly or via a linker. In some embodiments, thefluorescent label is attached to a phosphate group via a non-covalentlinker as described, e.g., in U.S. Pat. No. 8,252,910. In someembodiments, the linker is a hydrocarbon selected from substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted cycloalkyl, and substituted orunsubstituted heterocycloalkyl; see, e.g., U.S. Pat. No. 8,367,813. Thelinker may also comprise a nucleic acid strand; see, e.g., U.S. Pat. No.9,464,107.

In embodiments in which the fluorescent label is linked to a phosphategroup, the nucleotide precursor is incorporated into the nascent chainby the nucleic acid polymerase, which also cleaves and releases thedetectable fluorescent label. In some embodiments, the fluorescent labelis removed by cleaving the linker, e.g., as described in U.S. Pat. No.9,587,275.

In some embodiments, the nucleotide precursors are non-extendable“terminator” nucleotides, i.e., the nucleotides that have a 3′-endblocked from addition of the next nucleotide by a blocking “terminator”group. The blocking groups are reversible terminators that can beremoved in order to continue the strand synthesis process as describedherein. Attaching removable blocking groups to nucleotide precursors isknown in the art. See, e.g., U.S. Pat. Nos. 7,541,444, 8,071,739 andcontinuations and improvements thereof. Briefly, the blocking group maycomprise an allyl group that can be cleaved by reacting in aqueoussolution with a metal-allyl complex in the presence of phosphine ornitrogen-phosphine ligands. Other examples of reversible terminatornucleotides used in sequencing by synthesis include the modifiednucleotides described in U.S. Provisional App. Ser. No. 62/781,638 filedon Dec. 19, 2018 and entitled “3′-Protected Nucleotides,” which ishereby incorporated by reference in its entirety for all purposes.

In the embodiment illustrated in FIG. 2C, Image 1 is taken with thetemperature within a first temperature range, and Image 2 is taken withthe temperature within a second, lower temperature range. If the emittedintensity is below the threshold in both images, it is determined thatthe incorporated nucleotide is guanine (G). If the emitted intensity isabove the threshold in Image 2 but below the threshold in Image 1, it isdetermined that the incorporated nucleotide is cytosine (C). If theemitted intensity is above the threshold in both images, adenine (A) andthymine (T) can be distinguished by comparing the intensity of emittedlight in the two images. In the example of FIG. 2C, if the detectedintensity is approximately the same in the two images, it can bedetermined that the incorporated nucleotide is thymine (T), because thefirst dye emits light of a known intensity in both temperature ranges.It is to be understood that the intensity of emitted light might beapproximately the same in Images 1 and 2 when (in the example) thyminehas been incorporated. The intensity would be approximately the same,for example, if the dye IV of FIG. 1C is used to label thymine.Alternatively, the intensity associated with thymine could be differentin Image 1 and Image 2. For example, if the dye II of FIG. 1C were usedto label thymine, the measured intensity in the two images may bedifferent. It would, however, be known what intensity to expect from thedye labeling thymine at the respective temperatures at which Images 1and 2 are taken.

Because the third nucleotide (shown as adenine (A) in FIG. 2C) has beenlabeled with two dyes, one of which also labels thymine (T), and theintensity emitted by the dye labeling thymine is known for the twotemperatures at which Images 1 and 2 are taken, it can be determinedwhether adenine (A) or thymine (T) has been incorporated because theemitted intensity of Image 2 will be higher when the incorporatednucleotide is adenine (A) than when it is thymine (T). Thus, if thedetected intensity is higher in Image 2 than in Image 1, and it isgreater than the expected emitted intensity for the dye labeling thymine(T), it can be determined that the incorporated nucleotide is adenine(A). One advantage of this embodiment is that there is no need to addany chemical reagents to change or remove any labels between taking thetwo images. The incorporated nucleotide can be determined simply bycomparing the detected intensity levels in Images 1 and 2. The tablebelow provides the decision table associated with the example shown inFIG. 2C and described above, where threshold 1 is the intensitythreshold to detect the first dye in temperature Range 1; threshold 2,which may be the same as or different from threshold 1, is the intensitythreshold to detect the first dye in temperature Range 2; threshold 3 isthe intensity threshold to detect the second dye in temperature Range 2;and threshold 4, which is greater than both of thresholds 2 and 3, isthe intensity threshold to detect the presence of both the first andsecond dyes in temperature Range 2.

Image 1 intensity Image 2 intensity Determination <threshold 1<threshold 2 guanine (G) <threshold 1 ≥threshold 3 cytosine (C)≥threshold 1 ≥threshold 2 and <threshold 4 thymine (T) ≥threshold 1≥threshold 4 adenine (A)

One benefit of the approach shown in FIG. 2C and described above is thatit does not require any additional chemistry steps within a cycle,unlike conventional approaches using two imaging events.

FIG. 3A is a flowchart illustrating a method 100 of sequencing nucleicacid in accordance with some embodiments. The method 100 involves theuse of a sequencing apparatus comprising a fluidic channel having aplurality of sites for binding a plurality of molecules of a nucleicacid polymerase or a plurality of nucleic acid strands (e.g., asdescribed above and as is known in the art) to a surface of the fluidicchannel. At 102 the method begins. At 104, material is added to thefluidic channel. The material includes at least the plurality of nucleicacid strands, the plurality of molecules of the nucleic acid polymerase,a first fluorescently-labeled nucleotide precursor, and a secondfluorescently-labeled nucleotide precursor.

The materials added in step 104 may be added to the fluidic channel atthe same time or in one or more rounds of addition. For example, in someembodiments, molecules of a nucleic acid polymerase are first bound tothe surface of the fluidic channel at the sites, and then the remainingmaterials are added to the fluidic channel. In other embodiments,nucleic acid strands are first bound to the surface of the fluidicchannel at the sites, and then the remaining materials are added to thefluidic channel.

The first fluorescently-labeled nucleotide precursor comprises a firstfluorescent label (which may be cleavable). The intensity emitted by thefirst fluorescent label is greater than or equal to a first threshold ina first temperature range and also in a second temperature range that islower than and does not overlap the first temperature range. In otherwords, the first fluorescent label will emit light at an intensity of atleast the first threshold in both the first and second temperatureranges. The first and second temperature ranges can be adjacent (e.g.,the first temperature range is from 30 to 50 degrees Celsius and thesecond temperature range is from 10 to 30 degrees Celsius), or they canbe separated by a gap in temperatures (e.g., the first temperature rangeis from 40 to 50 degrees Celsius, and the second temperature range isfrom 20 to 35 degrees Celsius).

The second fluorescently-labeled nucleotide precursor comprises a secondfluorescent label (which may be cleavable). The intensity emitted by thesecond fluorescent label is less than a second threshold in the firsttemperature range and greater than or equal to the second threshold inthe second temperature range. In other words, considering only the firstand second temperature ranges, the second fluorescent label will emitlight at an intensity of at least the second threshold only in thesecond (lower) temperature range; in the first (higher) temperaturerange, the second fluorescent label will not emit light at an intensitythat meets or exceeds the second threshold. The first and secondthresholds may be the same, or they may be different. It is to beappreciated that the second fluorescent label may emit light at anintensity of at least the second threshold in temperature ranges outsideof the first and second temperature ranges. For example, the secondfluorescent label may emit light at an intensity of at least the secondthreshold at temperatures below the second temperature range. It is alsocontemplated that the second fluorescent label may emit light at anintensity of at least the second threshold at temperatures above thefirst temperature range.

There are a number of ways to attach and, if necessary, cleave thefluorescent labels. For example, the fluorescent labels may be attachedto a base, in which case they may be cleaved chemically. As anotherexample, the fluorescent labels may be attached to a phosphate, in whichcase they may be cleaved by polymerase or, if attached via a linker, bycleaving the linker.

In some embodiments, the fluorescent label is linked to the nitrogenousbase (A, C, T, G, or a derivative) of the nucleotide precursor. Afterincorporation of the nucleotide precursor and detection, the fluorescentlabel is cleaved from the incorporated nucleotide.

In some embodiments, the fluorescent label is attached via a cleavablelinker. Cleavable linkers are known in the art and have been described,e.g., in U.S. Pat. Nos. 7,057,026, 7,414,116 and continuations andimprovements thereof. In some embodiments, the fluorescent label isattached to the 5-position in pyrimidines or the 7-position in purinesvia a linker comprising an allyl or azido group. In other embodiments,the linker comprises a disulfide, indole, a Sieber group, a t-butylSieber group, or a dialkoxybenzyl group. The linker may further containone or more substituents selected from alkyl (C₁₋₆) or alkoxy (C₁₋₆),nitro, cyano, fluoro groups or groups with similar properties. Briefly,the linker can be cleaved by water-soluble phosphines or phosphine-basedtransition metal-containing catalysts. Other linkers and linker cleavagemechanisms are known in the art. For example, linkers comprising tritylgroups, p-alkoxybenzyl ester groups, p-alkoxybenzyl amide groups,tert-butyloxycarbonyl (Boc) groups, and acetal based groups can becleaved under acidic conditions by a proton-releasing cleavage agent,such as an acid. A thioacetal or other sulfur-containing linker can becleaved using a thiophilic metals, such as nickel, silver or mercury.The cleavage protecting groups can also be considered for thepreparation of suitable linker molecules. Ester- and disulfidecontaining linkers can be cleaved under reductive conditions. Linkerscontaining triisopropyl silane (TIPS) or t-butyldimethyl silane (TBDMS)can be cleaved in the presence of F ions. Photocleavable linkers cleavedby a wavelength that does not affect other components of the reactionmixture include linkers comprising o-nitrobenzyl groups. Linkerscomprising benzyloxycarbonyl groups can be cleaved by Pd-basedcatalysts.

In some embodiments, the nucleotide precursor comprises a label attachedto a polyphosphate moiety as described in, e.g., U.S. Pat. Nos.7,405,281 and 8,058,031. Briefly, the nucleotide precursor comprises anucleoside moiety and a chain of 3 or more phosphate groups where one ormore of the oxygen atoms are optionally substituted, e.g., with S. Thelabel may be attached to the α, β, γ or higher phosphate group (ifpresent) directly or via a linker. In some embodiments, the label isattached to a phosphate group via a non-covalent linker as described,e.g., in U.S. Pat. No. 8,252,910. In some embodiments, the linker is ahydrocarbon selected from substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted cycloalkyl, and substituted or unsubstitutedheterocycloalkyl; see, e.g., U.S. Pat. No. 8,367,813. The linker mayalso comprise a nucleic acid strand; see, e.g., U.S. Pat. No. 9,464,107.

In embodiments in which the fluorescent label is linked to a phosphategroup, the nucleotide precursor is incorporated into the nascent chainby the nucleic acid polymerase, which also cleaves and releases thedetectable fluorescent label. In some embodiments, the fluorescent labelis removed by cleaving the linker, e.g., as described in U.S. Pat. No.9,587,275.

In some embodiments, the nucleotide precursors are non-extendable“terminator” nucleotides, i.e., the nucleotides that have a 3′-endblocked from addition of the next nucleotide by a blocking “terminator”group. The blocking groups are reversible terminators that can beremoved in order to continue the strand synthesis process as describedherein. Attaching removable blocking groups to nucleotide precursors isknown in the art. See, e.g., U.S. Pat. Nos. 7,541,444, 8,071,739 andcontinuations and improvements thereof. Briefly, the blocking group maycomprise an allyl group that can be cleaved by reacting in aqueoussolution with a metal-allyl complex in the presence of phosphine ornitrogen-phosphine ligands. Other examples of reversible terminatornucleotides used in sequencing by synthesis are known in the art.

Referring again to FIG. 3A, at 106, after the nucleic acid strands,polymerase molecules, and first and second fluorescently-labelednucleotide precursors have been added to the fluidic channel, thetemperature of the contents of the fluidic channel is set to be withinthe first temperature range. At 108, the apparatus is illuminated (e.g.,by a laser) while the temperature is within the first temperature range,and a first image of the contents of the fluidic channel is captured,i.e., a first intensity at each of the plurality of sites is detectedwhile the temperature of the fluidic channel is within the firsttemperature range. The first image captures the intensity at each of theplurality of sites. As explained previously, an image is a record of thepresence or absence of emitted light (e.g., an optical response over aplurality of physical locations).

At 110, the image is assessed to determine whether the intensity of thefirst image at a particular site of the plurality of sites is greaterthan or equal to a first value. If the intensity of the first image atthe particular site is greater than or equal to the first value, then,at 112 it is determined that the first fluorescently-labeled nucleotideprecursor has been incorporated into the extendable primer at that site.The same approach can be used to determine, for some or all of the othersites, whether the first fluorescently-labeled nucleotide precursor hasbeen incorporated into the extendable primer at those sites.

If, at 110, it is determined that the intensity of the first image atthe particular site is not greater than or equal to the first value, at114 the temperature of the fluidic channel's contents is set to bewithin the second (lower) temperature range. At 116, the apparatus isilluminated (e.g., by a laser) while the temperature is within thesecond temperature range, and a second image of the contents of thefluidic channel is captured, i.e., a second intensity at each of theplurality of sites is detected while the temperature of the fluidicchannel is within the second temperature range. The second imagecaptures the intensity at each of the plurality of sites. At 118, it isdetermined whether the intensity of the second image at a particularsite of the plurality of sites is greater than or equal to a secondvalue and whether the intensity of the first image at the particularsite is less than the first value. The first and second values may beapproximately the same, or they may be different. In some embodiments,the first value is the first threshold, and the second value is thesecond threshold. In some embodiments, the first value is based on thefirst threshold, and the second value is based on the second threshold.For example, the first value may be a multiple of the first threshold ora percentage of the first threshold. Similarly, the second value may bemultiple of the second threshold or a percentage of the secondthreshold.

If the intensity of the second image at a particular site of theplurality of sites is greater than or equal to a second value and theintensity of the first image at the particular site is less than thefirst value, then at 120 it is determined that the secondfluorescently-labeled nucleotide precursor has been incorporated intothe extendable primer at that site. The same approach can be used todetermine, for some or all of the other sites, whether the secondfluorescently-labeled nucleotide precursor has been incorporated intothe extendable primer at those sites.

As explained above, the method 100 illustrated in FIG. 3A enables adetermination of whether either of the two fluorescently-labelednucleotide precursors has been incorporated during a cycle of anucleotide sequencing process. The steps 104 through 120 can beperformed again in one or more subsequent sequencing cycles, e.g., aftercleaving the first and second fluorescent labels.

As described in more detail below, in some embodiments, the method 100includes steps beyond those shown in FIG. 3A. For example, in someembodiments, the method 100 includes steps illustrated in FIG. 3B or thesteps illustrated in FIG. 3C.

It is to be appreciated that although FIG. 3A illustrates the method 100as if the temperature of the contents of the fluidic channel isinitially set to be within the highest temperature range and thencooled, an implementation could begin with the temperature in the secondtemperature range and then heat the contents of the fluidic chamber tobe within the first (higher) temperature range. Such an embodiment isshown in FIG. 3D. As shown, the steps associated with the firsttemperature range and the first image may take place after the stepsassociated with the second temperature range and the second image.

Referring again to FIG. 3A, in some embodiments the method 100terminates after 112 because it has been determined that the firstfluorescently-labeled nucleotide precursor has been incorporated at thesite. It is to be appreciated, however, that although FIG. 3A shows thesteps occurring in a particular order (e.g., step 110 occurring beforestep 114), various of the steps can occur in a different order thanshown. For example, as explained above in the context of FIG. 3D, thelower temperature range may be tested first. As another example, it isnot necessary to make the determination of whether the intensity isgreater than or equal to a specified value between imaging steps. Insome embodiments, the first and second images are captured, and then theimages are analyzed to determine whether the first or secondfluorescently-labeled nucleotide precursor was incorporated. FIG. 3Eillustrates such an embodiment. It is to be understood that althoughFIG. 3E illustrates steps 106 and 108 taking place before steps 114 and116, steps 114 and 116 may be performed before steps 106 and 108.

Referring again to FIG. 3A, if, at 118, it is determined that theintensity of the second image at the particular site is not greater thanor equal to the second value or the intensity of the first image at theparticular site is not less than the first value, then it is concludedthat neither the first nor the second fluorescently-labeled nucleotideprecursor has been incorporated at the particular site. In that case,the method 100 can end, or further analysis can be performed. FIG. 3Billustrates how the method 100 can continue in accordance with someembodiments. FIG. 3B shows the steps to determine whether at least athird fluorescently-labeled nucleotide precursor has been incorporatedat the particular site. FIG. 3B also provides for the determination ofwhether a fourth fluorescently-labeled nucleotide precursor has beenincorporated at the particular site. The combination of FIG. 3A and FIG.3B shows a method 100 of implementing the approach illustrated in FIG.2A, discussed above.

Referring to FIG. 3B, at 130, a third fluorescently-labeled nucleotideprecursor is added to the fluidic channel. Step 130 may be combined withstep 104 shown FIG. 3A (i.e., the first, second, and thirdfluorescently-labeled nucleotide precursors may be added to the fluidchamber at substantially the same time), or it may be a separate step.The third fluorescently-labeled nucleotide precursor comprises a thirdfluorescent label (which may be cleavable). The intensity emitted by thethird fluorescent label is less than a third threshold in the first andsecond temperature ranges and greater than or equal to the thirdthreshold in a third temperature range that is lower than both the firstand second temperature ranges. In other words, considering only thefirst, second, and third temperature ranges, the third fluorescent labelwill emit light at an intensity of at least the third threshold only inthe third temperature range; in the higher first and second temperatureranges, the third fluorescent label will not emit light at an intensitythat meets or exceeds the third threshold. The first, second, and thirdtemperature ranges are non-overlapping, and they may be adjacent to eachother, or there may be a temperature gap between the first and secondtemperature ranges and/or between the second and third temperatureranges. It is to be appreciated that the third fluorescent label mayemit light at an intensity of at least the third threshold intemperature ranges outside of the first, second, and third temperatureranges. For example, the third fluorescent label may emit light at anintensity of at least the third threshold at temperatures below thethird temperature range. It is also contemplated that the thirdfluorescent label may emit light at an intensity of at least the thirdthreshold at temperatures above the first temperature range. The thirdthreshold may be approximately the same as (i.e., equal to) the firstthreshold and/or the second threshold. Alternatively, the thirdthreshold may be different from the first threshold and/or the secondthreshold.

At 132, the temperature of the contents of the fluidic channel is set tobe within the third temperature range. At 134, the apparatus isilluminated while the temperature is within the third temperature range,and a third image of the contents of the fluidic channel is captured,i.e., a third intensity at each of the plurality of sites is detectedwhile the temperature of the fluidic channel is within the thirdtemperature range. The third image captures the intensity at each of theplurality of sites.

At 136, the third image is assessed to determine whether the intensityof the third image at a particular site of the plurality of sites isgreater than or equal to a third value. The third value may beapproximately the same as (or equal to) the first value and/or thesecond value. If the intensity of the third image at the particular siteis greater than or equal to the third value, and the intensity of thefirst image at the particular site is less than the first value, and theintensity of the second image at the particular site is less than thesecond value, then, at 138, it is determined that the thirdfluorescently-labeled nucleotide precursor has been incorporated intothe extendable primer at that site. The same approach can be used todetermine, for some or all of the other sites, whether the thirdfluorescently-labeled nucleotide precursor has been incorporated intothe extendable primer at those sites.

If, at 136, it is determined that the intensity of the third image atthe particular site is not greater than or equal to the third value, at140, a fourth fluorescently-labeled nucleotide precursor is added to thefluidic chamber. Step 140 may be combined with step 130 and/or step 104(shown in FIG. 3A). The fourth fluorescently-labeled nucleotideprecursor comprises a fourth fluorescent label (which may be cleavable).The intensity emitted by the fourth fluorescent label is less than afourth threshold in the first, second, and third temperature ranges andgreater than or equal to the fourth threshold in a fourth temperaturerange that is lower than all of the first, second, and third temperatureranges. In other words, considering only the first, second, third, andfourth temperature ranges, the fourth fluorescent label will emit lightat an intensity of at least the fourth threshold only in the fourthtemperature range; in the higher first, second, and third temperatureranges, the fourth fluorescent label will not emit light at an intensitythat meets or exceeds the fourth threshold. It is to be appreciated thatthe fourth fluorescent label may emit light at an intensity of at leastthe fourth threshold in temperature ranges outside of the first, second,third, and fourth temperature ranges. For example, the fourthfluorescent label may emit light at an intensity of at least the fourththreshold at temperatures below the fourth temperature range. It is alsocontemplated that the fourth fluorescent label may emit light at anintensity of at least the fourth threshold at temperatures above thefirst temperature range. The first, second, third, and fourthtemperature ranges are non-overlapping, and they may be adjacent to eachother, or there may be a temperature gap between the first and secondtemperature ranges, between the second and third temperature ranges,and/or between the third and fourth temperature ranges. The fourththreshold may be approximately the same as (i.e., equal to) the firstthreshold, the second threshold, and/or the third threshold, or it maybe different from the first, second, and/or third thresholds.

At 142, the temperature of the fluidic channel's contents is set to bewithin the fourth temperature range. At 144, the apparatus isilluminated while the temperature is within the fourth temperaturerange, and a fourth image of the contents of the fluidic channel iscaptured, i.e., a fourth intensity at each of the plurality of sites isdetected while the temperature of the fluidic channel is within thefourth temperature range. The fourth image captures the intensity ateach of the plurality of sites. At 146, it is determined whether theintensity of the fourth image at a particular site of the plurality ofsites is greater than or equal to a fourth value. The fourth value maybe approximately the same as (i.e., equal to) the first value, thesecond value, and/or third value. If so, and if the intensity of thefirst image at the particular site is less than the first value, and theintensity of the second image at the particular site is less than thesecond value, and the intensity of the third image at the particularsite is less than the third value, then, at 148 it is determined thatthe fourth fluorescently-labeled nucleotide precursor has beenincorporated into the extendable primer at that site. The same approachcan be used to determine, for some or all of the other sites, whetherthe fourth fluorescently-labeled nucleotide precursor has beenincorporated into the extendable primer at those sites.

It is to be understood that FIG. 3D or FIG. 3E can be substituted forFIG. 3A. Likewise, it is to be understood that changes similar to thosedescribed above in the context of FIGS. 3D and 3E can be made to thecombination of FIG. 3A and FIG. 3B. Specifically, the order in which thetemperature ranges are tested need not be from highest to lowest, oreven monotonic (i.e., it is contemplated that the temperature ranges maybe tested in any order). Similarly, it is not necessary to analyze eachimage after it is taken and before changing the temperature of thecontents of the fluidic channel (i.e., two or more images may be takenprior to any analysis). In addition, and as explained above, when morethan two nucleotide precursors are to be added to the fluidic chamber,they may be added in one or more rounds of addition (e.g., all materialscan be added in step 104, or two or more of steps 104, 130, and 140 canbe combined).

As explained above in the context of FIG. 2B, some embodiments use onlythree images to enable determination of which of four nucleotideprecursors has been incorporated at a particular site. The combinationof FIGS. 3A and 3C illustrates the method 100 in accordance with somesuch embodiments. After step 118 (FIG. 3A), the method 100 continueswith step 160, in which a third fluorescently-labeled nucleotideprecursor and a fourth, unlabeled nucleotide precursor are added to thefluidic channel. Step 160 may be combined with step 104 (FIG. 3A), or itmay be a separate step. The third fluorescently-labeled nucleotideprecursor comprises the third fluorescent label, as described above. Thefourth nucleotide precursor is unlabeled.

At 162, the temperature of the contents of the fluidic channel is set tobe within the third temperature range. At 164, the apparatus isilluminated while the temperature is within the third temperature range,and a third image of the contents of the fluidic channel is captured,i.e., a third intensity at each of the plurality of sites is detectedwhile the temperature of the fluidic channel is within the thirdtemperature range. The third image captures the intensity at each of theplurality of sites.

At 166, the third image is assessed to determine whether the intensityof the third image at a particular site of the plurality of sites isgreater than or equal to a third value. The third value may beapproximately the same as (i.e., equal to) the first value and/or thesecond value, or it may be different. If the intensity of the thirdimage at the particular site is greater than or equal to the thirdvalue, and the intensity of the first image at the particular site isless than the first value, and the intensity of the second image at theparticular site is less than the second value, then, at 168, it isdetermined that the third fluorescently-labeled nucleotide precursor hasbeen incorporated into the extendable primer at that site. The sameapproach can be used to determine, for some or all of the other sites,whether the third fluorescently-labeled nucleotide precursor has beenincorporated into the extendable primer at those sites.

If, at 166, it is determined that the intensity of the third image atthe particular site is not greater than or equal to the third value, at170, it is inferred that the fourth, unlabeled nucleotide precursor hasbeen incorporated at the particular site.

As explained above in the context of FIG. 2C, some embodiments use onlytwo images to enable determination of which of four nucleotideprecursors has been incorporated at a particular site. FIGS. 4A and 4Billustrate a method 200 in accordance with some such embodiments.

At 202, in one or more rounds of addition, material is added to thefluidic channel of a sequencing apparatus. The material includes atleast a plurality of nucleic acid strands, a plurality of molecules ofnucleic acid polymerase, a first fluorescently-labeled nucleotideprecursor, a second fluorescently-labeled nucleotide precursor, a thirdfluorescently-labeled nucleotide precursor, and a fourth, unlabelednucleotide precursor. The nucleic acid strands and nucleic acidpolymerase were previously described in the context of FIG. 3A, andthose descriptions are also applicable here. In addition, it waspreviously explained how the molecules of the nucleic acid polymerase orthe plurality of nucleic acid strands can be bound to a surface of thefluidic channel of the sequencing apparatus. Those explanations applyhere, too.

The first fluorescently-labeled nucleotide precursor comprises a firstfluorescent label. The intensity emitted by the first fluorescent labelis less than a first threshold in a first temperature range and greaterthan or equal to the first threshold in a second temperature range thatis lower than the first temperature range. In other words, the firstfluorescent label will emit light at an intensity of at least the firstthreshold in the second temperature range, but not in the firsttemperature range. The label need not be cleavable. It is to beappreciated that the first fluorescent label may emit light at anintensity of at least the first threshold in temperature ranges outsideof the first and second temperature ranges. For example, the firstfluorescent label may emit light at an intensity of at least the firstthreshold at temperatures below the second temperature range. It is alsocontemplated that the first fluorescent label may emit light at anintensity of at least the first threshold at temperatures above thefirst temperature range.

The second fluorescently-labeled nucleotide precursor comprises a secondfluorescent label (which need not be cleavable). The intensity emittedby the second fluorescent label is greater than or equal to a secondthreshold in the first temperature range and in the second temperaturerange. In other words, considering only the first and second temperatureranges, the second fluorescent label will emit light at an intensity ofat least the second threshold in both the first and second temperatureranges. The second threshold may be approximately the same as (i.e.,equal to) the first threshold, or the two thresholds may be different.

The third fluorescently-labeled nucleotide precursor comprises both thefirst and second fluorescent labels.

The fourth nucleotide precursor does not include a fluorescent label.

There are a number of ways to attach the fluorescent labels to the firstand second fluorescently-labeled nucleotide precursors, as describedabove in the context of FIG. 3A.

Referring again to FIG. 4A, at 204, the temperature of the contents ofthe fluidic channel is set to be within the first temperature range. At206, the apparatus is illuminated while the temperature is within thefirst temperature range, and a first image of the contents of thefluidic channel is captured, i.e., a first intensity at each of theplurality of sites is detected while the temperature of the fluidicchannel is within the first temperature range. The first image capturesthe intensity at each of the plurality of sites. At 208, the temperatureof the contents of the fluidic channel is set to be within the secondtemperature range. At 210, the apparatus is illuminated while thetemperature is within the second temperature range, and a second imageof the contents of the fluidic channel is captured, i.e., a secondintensity at each of the plurality of sites is detected while thetemperature of the fluidic channel is within the second temperaturerange. The second image captures the intensity at each of the pluralityof sites.

At 212, it is determined, using the first and second images, which ofthe first, second, third, or fourth nucleotide precursors has beenincorporated at a particular site of the plurality of sites. FIG. 4Billustrates the step 212 in accordance with some embodiments. At 220, itis determined whether the intensity of the first image at the particularsite is less than a first value and the intensity of the second image atthe particular site is greater than or equal to a second value. Thefirst and second values may be approximately the same (i.e., equal), orthey may be different. The first and second values may be, respectively,equal to the first and second thresholds. Alternatively, the first andsecond values may be, respectively, based on the first and secondthresholds (e.g., they may be multiples of or percentages of the firstand second thresholds). If the intensity of the first image at theparticular site is less than the first value and the intensity of thesecond image at the particular site is greater than or equal to thesecond value, then at 222 it is determined that the firstfluorescently-labeled nucleotide precursor has been incorporated at theparticular site. If not, then at 224 it is determined whether theintensity of the first image at the particular site is greater than orequal to the first value and the intensity of the second image at theparticular site is greater than or equal to a third value, which isgreater than each of the first and second values. The third value maybe, for example, the sum of the first and second values. If theintensity of the first image at the particular site is greater than orequal to the first value and the intensity of the second image at theparticular site is greater than or equal to the third value, then at 226it is determined that the third fluorescently-labeled nucleotideprecursor has been incorporated at the particular site. If not, then at228 it is determined whether the intensity of the first image at theparticular site is greater than or equal to the first value, and theintensity of the second image at the particular site is greater than orequal to the second value but less than the third value. If so, then at230, it is determined that the second fluorescently-labeled nucleotideprecursor has been incorporated at the particular site.

If it is not determined, at 228, that the intensity of the first imageat the particular site is greater than or equal to the first value, andthe intensity of the second image at the particular site is greater thanor equal to the second value but less than the third value, then it canbe inferred, at 234, that the fourth, unlabeled nucleotide precursor hasbeen incorporated at the particular site. Alternatively, and optionally,it can be positively determined, at step 232, whether the intensity ofthe first image at the particular site is less than the first value, andthe intensity of the second image at the particular site is less thanthe second value, in which case it is then determined, at 234, that thefourth, unlabeled nucleotide precursor has been incorporated at theparticular site.

The explanations of FIGS. 3A-3E and FIGS. 4A and 4B presume that theemitted intensities of the fluorescent labels have characteristics andintensity profiles similar to those generally illustrated elsewhere inthis document (e.g., in FIGS. 1A-1C and FIGS. 2A-2C). It is to beappreciated that it may be appropriate to modify certain of the elementsof FIGS. 3A-3E (e.g., 110, 118, 136, 146, 166) and/or FIGS. 4A and 4B(e.g., 220, 224, 228, 232) if the intensities of the fluorescent labelshave different characteristics (e.g., if they are non-monotonic withtemperature). Those having ordinary skill in the art will understand, inview of the disclosures herein, what modifications are appropriate tomake.

FIG. 5 illustrates a system 500 for sequencing nucleic acid. The system500 includes, as described previously, a fluidic channel 515 that has aplurality of sites for attaching, to a surface of the fluidic channel515, a plurality of nucleic acid strands to be sequenced. For example,the fluidic channel 515 may include a structure (e.g., a cavity or aridge) configured to anchor nucleic acid or a nucleic acid polymerase tothe proximal wall. A heater 510 is coupled to the fluidic channel 515.The heater 510 sets the temperature of the contents of the fluidicchannel 515. It is to be understood that the heater 510 may includecooling elements as well as or instead of heating elements. The primarycharacteristic of the heater 510 is that it is capable of controllingthe temperature of the contents of the fluidic channel 515. For example,the heater 510 is capable of setting the temperature of the contents ofthe fluidic channel 515 to be within at least any of nonoverlappingfirst, second, third, and fourth temperature ranges. The secondtemperature range may be lower than the first temperature range, thethird temperature range may be lower than the second temperature range,and the fourth temperature range may be lower than the third temperaturerange. The heater 510 may include, for example, thermal sensors, amicroprocessor, or software.

The system 500 also includes an imaging system 520. The imaging system520 is configured to detect the intensity at each of the plurality ofsites of the fluidic channel 515. For example, the imaging system 520 isable to detect the intensity at each of the plurality of sites in eachof the first, second, third, and fourth temperature ranges.

As illustrated in FIG. 6, the imaging system 520 may comprise, forexample, some or all of: a camera 521, an excitation light source 522(e.g., a laser), illumination optics 523 (e.g., configured to uniformlydistribute the excitation light and reduce stray light), a detector 524(e.g., a lens having a large aperture to increase light collectingefficiency), an optical blocking filter 525 (e.g., to isolate thedetector 524 from the excitation light), an analog-to-digital converter(ADC), and/or one or more sensors 526 to translate the detectedintensity into a digitized signal or value (e.g., charge-coupled device(CCD) sensors, which may themselves include other components, such asADCs). It is to be understood that the components and elements listedabove are merely examples and are not intended to be limiting. Theimaging system 520 may be coupled to a storage device (not shown). Insuch embodiments, the storage device may store intensity data collectedby the imaging system 520.

The system 500 also includes at least one processor 505 coupled to theimaging system 520 and to the heater 510. The processor 505 executesmachine-readable instructions. The processor 505 may direct or controlother elements of the system 500. For example, the processor 505 maydirect the heater 510 to set the temperature of the contents of thefluidic channel 515 to be within a specified range. Similarly, theprocessor 505 may direct the imaging system 520 to detect the intensityat all or a subset of the plurality of sites of the fluidic channel 515.In addition or alternatively, the processor 505 may obtain an indicationof the detected intensity at all or a subset of the plurality of sitesof the fluidic channel 515 from the imaging system.

For example, in response to the intensity at a particular site of theplurality of sites being greater than or equal to a first threshold inthe first temperature range and in the second temperature range, theprocessor 505 may identify a first fluorescently-labeled nucleotideprecursor or a complementary base of the first fluorescently-labelednucleotide precursor. For example, if the processor 505 determines thatthe intensity at the particular site corresponds to the fluorescentlabel attached to a guanine precursor, the processor 505 may identifyguanine as the detected label, or cytosine as the next base in thefragment of nucleic acid being sequenced.

Similarly, in response to the intensity at the particular site of theplurality of sites being less than a second threshold in the firsttemperature range and greater than or equal to the second threshold inthe second temperature range, the processor 505 may identify a secondfluorescently-labeled nucleotide precursor or a complementary base ofthe second fluorescently-labeled nucleotide precursor. Likewise, inresponse to the intensity at the particular site of the plurality ofsites being less than a third threshold in the first and secondtemperature ranges and greater than or equal to the third threshold inthe third temperature range, the processor 505 may identify a thirdfluorescently-labeled nucleotide precursor or a complementary base ofthe third fluorescently-labeled nucleotide precursor. And, finally, inresponse to the intensity at the particular site of the plurality ofsites being less than a fourth threshold in each of the first, second,and third temperature ranges and greater than or equal to a fourththreshold in the fourth temperature range, the processor 505 mayidentify a fourth fluorescently-labeled nucleotide precursor or acomplementary base of the fourth fluorescently-labeled nucleotideprecursor.

The processor 505 may instruct the heater 510 to set the temperature ofthe contents within the fluidic channel to be within the firsttemperature range. The processor 505 may then obtain, from the imagingsystem 520, an indication of a first intensity at some or all of theplurality of sites while the temperature of the contents of the fluidicchannel 520 is within the first temperature range. The processor 505 mayinstruct the heater 510 to set the temperature within the fluidicchannel to be within the second temperature range. The processor 505 maythen obtain, from the imaging system 520, an indication of a secondintensity at some or all of the plurality of sites while the temperatureof the fluidic channel 515 is within the second temperature range. Inresponse to the first intensity at a particular site of the plurality ofsites being less than a first value, and the second intensity at theparticular site being greater than or equal to a second value, theprocessor 505 may determine that a first fluorescently-labelednucleotide precursor has been incorporated into an extendable primer atthe particular site. For example, if the processor 505 determines thatthe intensity at the particular site corresponds to the fluorescentlabel attached to an adenine precursor, the processor 505 may identifyadenine as the detected label, or thymine as the next base in thefragment of nucleic acid being sequenced. Alternatively, in response tothe first intensity at the particular site being greater than or equalto the first value, and the second intensity at the particular sitebeing greater than or equal to the second value and less than a thirdvalue, the third value being greater than each of the first and secondvalues, the processor 505 may determine that a secondfluorescently-labeled nucleotide precursor has been incorporated intothe extendable primer at the particular site. Finally, in response tothe first intensity at the particular site being greater than or equalto the first value, and the second intensity at the particular sitebeing greater than or equal to the third value, the processor 505 maydetermine that a third fluorescently-labeled nucleotide precursor hasbeen incorporated into the extendable primer at the particular site.

In the foregoing description and in the accompanying drawings, specificterminology has been set forth to provide a thorough understanding ofthe disclosed embodiments. In some instances, the terminology ordrawings may imply specific details that are not required to practicethe invention.

To avoid obscuring the present disclosure unnecessarily, well-knowncomponents are shown in block diagram form and/or are not discussed indetail or, in some cases, at all.

Unless otherwise specifically defined herein, all terms are to be giventheir broadest possible interpretation, including meanings implied fromthe specification and drawings and meanings understood by those skilledin the art and/or as defined in dictionaries, treatises, etc. As setforth explicitly herein, some terms may not comport with their ordinaryor customary meanings.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” do not exclude plural referents unless otherwisespecified. The word “or” is to be interpreted as inclusive unlessotherwise specified. Thus, the phrase “A or B” is to be interpreted asmeaning all of the following: “both A and B,” “A but not B,” and “B butnot A.” Any use of “and/or” herein does not mean that the word “or”alone connotes exclusivity.

As used in the specification and the appended claims, phrases of theform “at least one of A, B, and C,” “at least one of A, B, or C,” “oneor more of A, B, or C,” and “one or more of A, B, and C” areinterchangeable, and each encompasses all of the following meanings: “Aonly,” “B only,” “C only,” “A and B but not C,” “A and C but not B,” “Band C but not A,” and “all of A, B, and C.”

To the extent that the terms “include(s),” “having,” “has,” “with,” andvariants thereof are used in the detailed description or the claims,such terms are intended to be inclusive in a manner similar to the term“comprising,” i.e., meaning “including but not limited to.” The terms“exemplary” and “embodiment” are used to express examples, notpreferences or requirements.

The drawings are not necessarily to scale, and the dimensions, shapes,and sizes of the features may differ substantially from how they aredepicted in the drawings.

Although specific embodiments have been disclosed, it will be evidentthat various modifications and changes may be made thereto withoutdeparting from the broader spirit and scope of the disclosure. Forexample, features or aspects of any of the embodiments may be applied,at least where practicable, in combination with any other of theembodiments or in place of counterpart features or aspects thereof.Accordingly, the specification and drawings are to be regarded in anillustrative rather than a restrictive sense.

We claim:
 1. A method of sequencing nucleic acid using a sequencingapparatus comprising a fluidic channel having a plurality of sites forattaching, to a surface of the fluidic channel, a plurality of nucleicacid strands to be sequenced, the method comprising: in one or morerounds of addition, adding, to the fluidic channel, (i) the plurality ofnucleic acid strands, (ii) a plurality of molecules of nucleic acidpolymerase, (iii) a first fluorescently-labeled nucleotide precursorcomprising a first fluorescent label, wherein, when excited intofluorescence, an intensity of light emitted by the first fluorescentlabel is greater than or equal to a first threshold in a firsttemperature range and in a second temperature range, the secondtemperature range being lower than the first temperature range, and (iv)a second fluorescently-labeled nucleotide precursor comprising a secondfluorescent label, wherein, when excited into fluorescence, an intensityof light emitted by the second fluorescent label is less than or equalto a second threshold in the first temperature range and greater than orequal to the second threshold in the second temperature range; setting atemperature within the fluidic channel to be within the firsttemperature range; detecting a first intensity at each of the pluralityof sites while the temperature of the fluidic channel is within thefirst temperature range; in response to the first intensity at aparticular site of the plurality of sites being greater than or equal toa first value, determining that the first fluorescently-labelednucleotide precursor has been incorporated into an extendable primer atthe particular site; setting the temperature within the fluidic channelto be within the second temperature range; detecting a second intensityat each of the plurality of sites while the temperature of the fluidicchannel is within the second temperature range; and in response to thesecond intensity at the particular site being greater than or equal to asecond value and the first intensity at the particular site being lessthan the first value, determining that the second fluorescently-labelednucleotide precursor has been incorporated into the extendable primer atthe particular site.
 2. The method of claim 1, wherein the first andsecond thresholds are approximately the same.
 3. The method of claim 1,wherein the first and second thresholds are different.
 4. The method ofclaim 1, wherein the first and second values are approximately the same.5. The method of claim 1, wherein the first and second values aredifferent.
 6. The method of claim 1, wherein: the first value is thefirst threshold, and the second value is the second threshold.
 7. Themethod of claim 1, wherein: the first value is based on the firstthreshold, and the second value is based on the second threshold.
 8. Themethod of claim 1, further comprising: adding, to the fluidic channel, athird fluorescently-labeled nucleotide precursor comprising a thirdfluorescent label, wherein, when excited into fluorescence, an intensityof light emitted by the third fluorescent label is less than a thirdthreshold in the first and second temperature ranges and greater than orequal to the third threshold in a third temperature range, the thirdtemperature range being lower than the second temperature range; settingthe temperature within the fluidic channel to be within the thirdtemperature range; detecting a third intensity at each of the pluralityof sites while the temperature of the fluidic channel is within thethird temperature range; and in response to the third intensity at theparticular site being greater than or equal to a third value, and thefirst intensity at the particular site being less than the first value,and the second intensity at the particular site being less than thesecond value, determining that the third fluorescently-labelednucleotide precursor has been incorporated into the extendable primer atthe particular site.
 9. The method of claim 8, wherein the first,second, and third fluorescently-labeled nucleotide precursors are addedto the fluidic channel at substantially the same time.
 10. The method ofclaim 8, further comprising: in response to the first intensity at theparticular site being less than the first value, and the secondintensity at the particular site being less than the second value, andthe third intensity at the particular site being less than the thirdvalue, determining that a fourth, unlabeled precursor has beenincorporated into the extendable primer at the particular site.
 11. Themethod of claim 10, wherein at least two of the first, second, and thirdthresholds are approximately the same.
 12. The method of claim 10,wherein the first and second thresholds are different.
 13. The method ofclaim 10, wherein at least two of the first, second, and third valuesare approximately the same.
 14. The method of claim 10, wherein thefirst, second, and third values are different.
 15. The method of claim8, further comprising: adding, to the fluidic channel, a fourthfluorescently-labeled nucleotide precursor comprising a fourthfluorescent label, wherein, when excited into fluorescence, an intensityof light emitted by the fourth fluorescent label is less than a fourththreshold in each of the first, second, and third temperature ranges andgreater than or equal to the fourth threshold in a fourth temperaturerange, the fourth temperature range being lower than the thirdtemperature range; setting the temperature within the fluidic channel tobe within the fourth temperature range; detecting a fourth intensity ateach of the plurality of sites while the temperature of the fluidicchannel is within the fourth temperature range; and in response to thefourth intensity at the particular site being greater than or equal to afourth value, and the first intensity at the particular site being lessthan the first value, and the second intensity at the particular sitebeing less than the second value, and the third intensity at theparticular site being less than the third value, determining that thefourth fluorescently-labeled nucleotide precursor has been incorporatedinto the extendable primer at the particular site.
 16. The method ofclaim 15, wherein two or more of the first, second, third, and fourththresholds are approximately the same.
 17. The method of claim 15,wherein two or more of the first, second, third, and fourth values areapproximately the same.
 18. The method of claim 15, wherein the first,second, third, and fourth fluorescently-labeled nucleotide precursorsare added to the fluidic channel at substantially the same time. 19-31.(canceled)
 32. A system for sequencing nucleic acid, comprising: afluidic channel having a plurality of sites for attaching, to a surfaceof the fluidic channel, a plurality of nucleic acid strands to besequenced; a heater coupled to the fluidic channel for setting atemperature of a contents of the fluidic channel to be within any offirst, second, third, and fourth temperature ranges, wherein the first,second, third, and fourth temperature ranges are nonoverlapping; animaging system configured to detect an intensity of light emitted ateach of the plurality of sites in each of the first, second, third, andfourth temperature ranges; and at least one processor coupled to theimaging system and to the heater and configured to execute at least onemachine-readable instruction that, when executed, causes the at leastone processor to: in response to an intensity of light emitted at aparticular site of the plurality of sites being greater than or equal toa first threshold in the first temperature range and in the secondtemperature range, identify a first fluorescently-labeled nucleotideprecursor or a complementary base of the first fluorescently-labelednucleotide precursor, in response to the intensity of light emitted atthe particular site of the plurality of sites being less than a secondthreshold in the first temperature range and greater than or equal tothe second threshold in the second temperature range, identify a secondfluorescently-labeled nucleotide precursor or a complementary base ofthe second fluorescently-labeled nucleotide precursor, in response tothe intensity of light emitted at the particular site of the pluralityof sites being less than a third threshold in the first and secondtemperature ranges and greater than or equal to the third threshold inthe third temperature range, identify a third fluorescently-labelednucleotide precursor or a complementary base of the thirdfluorescently-labeled nucleotide precursor, and in response to theintensity of light emitted at the particular site of the plurality ofsites being less than a fourth threshold in each of the first, second,and third temperature ranges and greater than or equal to a fourththreshold in the fourth temperature range, identify a fourthfluorescently-labeled nucleotide precursor or a complementary base ofthe fourth fluorescently-labeled nucleotide precursor. 33-44. (canceled)45. A system for sequencing nucleic acid, comprising: a fluidic channelhaving a plurality of sites for attaching, to a surface of the fluidicchannel, a plurality of nucleic acid strands to be sequenced; a heaterfor setting a temperature of a contents of the fluidic channel to bewithin either of first and second temperature ranges, wherein the firstand second temperature ranges are nonoverlapping; an imaging systemconfigured to detect an intensity of light emitted at each of theplurality of sites in each of the first and second temperature ranges;and at least one processor coupled to the imaging system and configuredto execute at least one machine-readable instruction that, whenexecuted, causes the at least one processor to: instruct the heater toset a temperature within the fluidic channel to be within the firsttemperature range; obtain, from the imaging system, an indication of afirst intensity of light emitted at each of the plurality of sites whilethe temperature of the fluidic channel is within the first temperaturerange; instruct the heater to set the temperature within the fluidicchannel to be within the second temperature range; obtain, from theimaging system, an indication of a second intensity of light emitted ateach of the plurality of sites while the temperature of the fluidicchannel is within the second temperature range; in response to the firstintensity of light emitted at a particular site of the plurality ofsites being less than a first value, and the second intensity of lightemitted at the particular site being greater than or equal to a secondvalue, determine that a first fluorescently-labeled nucleotide precursorhas been incorporated into an extendable primer at the particular site,in response to the first intensity of light emitted at the particularsite being greater than or equal to the first value, and the secondintensity of light emitted at the particular site being greater than orequal to the second value and less than a third value, the third valuebeing greater than each of the first and second values, determine that asecond fluorescently-labeled nucleotide precursor has been incorporatedinto the extendable primer at the particular site, and in response tothe first intensity of light emitted at the particular site beinggreater than or equal to the first value, and the second intensity oflight emitted at the particular site being greater than or equal to thethird value, determine that a third fluorescently-labeled nucleotideprecursor has been incorporated into the extendable primer at theparticular site.